番茄潜叶蛾CYP4M131基因克隆及功能分析

Cloning and functional analysis of CYP4M131 in tomato leafminer Tuta absoluta

中文关键词:番茄潜叶蛾  细胞色素P450解毒酶  杀虫剂  RNA干扰  表达谱  功能

keywords:Tuta absoluta  cytochrome P450  insecticide  RNA interference  expression profile  function

中文摘要:为有效防控番茄潜叶蛾Tuta absoluta，利用PCR技术结合cDNA末端快速扩增克隆番茄潜叶蛾体内细胞色素P450解毒酶系基因CYP4M131，对其进行生物信息学分析，采用实时荧光定量PCR技术分析其表达谱，利用RNA干扰技术解析其功能。结果显示：番茄潜叶蛾CYP4M131开放阅读框为1 518 bp，编码505个氨基酸残基，CYP4M131氨基酸序列与马铃薯块茎蛾Phthorimaea operculella的氨基酸序列聚为一支。CYP4M131在番茄潜叶蛾所有发育阶段及头、体壁、中肠、脂肪体4种组织中均有表达，其中在蛹期和中肠中的相对表达量最高。注射dsCYP4M131后7 h和96 h，番茄潜叶蛾体内CYP4M131的沉默效率分别为55.6%和86.2%，且番茄潜叶蛾在蜕皮化蛹过程中出现蜷缩、腹部凹陷等表型。注射dsCYP4M131后，番茄潜叶蛾幼虫死亡率显著增加，注射后16 d的死亡率高达88.7%，蛹重仅为1.51 mg，化蛹率和羽化率分别为48.8%和23.8%，显著低于对照。注射dsCYP4M131后，乙基多杀菌素LC₅₀处理的番茄潜叶蛾3龄幼虫死亡率比对照显著增加。表明CYP4M131参与调控番茄潜叶蛾的化蛹过程和其对乙基多杀菌素的代谢。

Abstract:To enhance the control of tomato leafminer Tuta absoluta, the cytochrome P450 detoxification enzyme gene CYP4M131 was cloned for the first time using PCR technology combined with rapid amplification of cDNA ends. Bioinformatics analysis was conducted, and its expression profile was analyzed through quantitative real-time PCR. Additionally, RNA interference technology was employed to investigate its function. The results showed that the open reading frame of CYP4M131 in T. absoluta was 1 518 bp, encoding 505 amino acid residues. Phylogenetic analysis showed that CYP4M131 clustered closely with the amino acid sequence of Phthorimaea operculella. Expression profiling demonstrated that CYP4M131 was expressed across all developmental stages and four different tissues (head, integument, midgut and fat body), with the highest relative expression observed in the pupal stage and midgut. After injection with dsCYP4M131, silencing efficiencies of CYP4M131 at seven hours and 96 h were 55.6% and 86.2%, respectively. Knockdown of CYP4M131 resulted in phenotypic abnormalities during pupation. Injection of dsCYP4M131 significantly increased the larval mortality, reaching 88.7% at day 16 post-injection. Additionally, the pupal weight was reduced to 1.51 mg, while and the pupation and emergence rates declined to 48.8% and 23.8%, respectively, significantly lower than those in the control group. Furthermore, the mortality rate of 3rd-instar larvae of T. absoluta treated with LC₅₀ spinetoram significantly increased following dsCYP4M131 injection, compared to the control. These findings indicate that CYP4M131 might be involved in regulating the pupation process of T. absoluta and its metabolic response to spinetoram.